Novel method for the selection of specific affinity binders by homogeneous noncompetitive assay

ABSTRACT

The invention generally relates to the field of immunochemistry including antibody therapy, diagnostics, and basic research and specifically relates to the area of selecting affinity molecules such as natural antibodies, including artificial antibodies, antibody mimics, and aptamers. The invention relates particularly to a method of selecting affinity molecules using a homogeneous noncompetitive assay in a high throughput process.

This application claims priority to U.S. Provisional No. 61/244,770filed Sep. 22, 2009, which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The invention generally relates to the field of immunochemistryincluding antibody therapy, diagnostics, and basic research andspecifically relates to the area of selecting affinity molecules such asnatural antibodies, including artificial antibodies, antibody mimics,and aptamers. The invention relates particularly to a method ofselecting affinity molecules using a homogeneous noncompetitive assay ina high throughput process.

BACKGROUND OF THE INVENTION

Antibodies and specific alternatives are a standard tool for researchproduct, diagnostic, and therapeutic applications. Discovery andcharacterization of affinity reagents for these applications can bechallenging and arduous, involving antigen preparation, in vitro and/orin vivo development of binders, as well as screening and isolation ofthose binders. For example, mouse monoclonal antibodies are generated byimmunization of mice with a purified antigen, to allow in vivodevelopment of IgG antibodies by the B cells, and selection of anappropriate antibody by screening the expression of hybridomas (Köhler &Milstein Nature (1975) 256(5517):495-7). More recently, antibodyfragments (e.g., single chain variable fragments (scFv), and V_(II)Hdomains) and artificial affinity binders (e.g., Affibodies, Monobodies,and DARPins) have been created and are developed by screening large genelibraries of potential binders with various panning technologies. Thesetechnologies have allowed the development of numerous protein scaffoldswith unique affinity interaction domains that bind target epitopes.

A plethora of affinity molecule panning/screening technologies have beendeveloped over the past decade and all share the requisite associationof expressed protein with its nucleic acid coding sequence, which servesto identify the affinity binder. These technologies can be generallydivided into two groups: in vivo and in vitro display. In vivotechnologies are based on the introduction by viral infection orcellular transfection of a single gene into a cell, expression of theaffinity binder protein from the gene, delivery of the binder to thesurface of the cell or phage, selection of the affinity molecule to animmobilized target molecule, and identification of the gene associatedwith the affinity binder (Hoogenboom, H. R. (2005) Nature Biotech 23(9),1105-16). Examples of in vivo type display technologies are bacterial,yeast, mammalian, insect, and phage display.

In vitro technologies use the basic protein expression apparatus of acell, either as a cell extract or a purified system (Shimizu, et al.Nature Biotechnology (2001) 19, 751-755), but do not require a viablecell to express the affinity binder. Therefore, the required associationof coding sequence with affinity binder is through a physical bond. Forribosome display, this is done by “freezing” the ribosome at the end(stop codon) of an mRNA transcript after it has completed translatingthe transcript into a protein, which is also bound to the ribosome(Hanes, J. et al. (1998) Proc Natl Acad Sci USA 95(24), 14130-5).Affinity molecules to the target molecule are selected similarly to invivo display technologies (i.e., with an immobilized target molecule)and the mRNA transcript reverse transcribed into DNA for amplification,identification, and cloning. RNA display covalently links the 3′ end ofan mRNA transcript to the translated affinity molecule protein using alinker, which is added to the 3′ ends of the mRNA and incorporated intothe affinity binder protein at its C-terminal end (Roberts, R. W., andSzostak, J. W. (1997) PNAS 94(23), 12297-302). DNA display physicallyassociates the affinity molecule to the DNA coding sequence, eitherusing a DNA replication initiator protein (RepA) fused to the affinitybinder (ref) or a Hae III DNA methyltransferase that specificallyrecognizes methylated sequences (Bertschinger & Neri Protein (2004) Eng.Des. Sel. 17(9), 699-707). While the former can be performed insolution, the latter requires individual reactions for each proteinexpression event using in vitro compartmentalization.

In vitro compartmentalization (IVC) was developed in 1998 by AndrewGriffiths and Dan Tawfik (Nature Biotech. 16, 652) as an alternative tostandard reaction vessels. Using cellular compartmentalization as amodel, this technology facilitates the creation of minuscule aqueoussolutions using water-in-oil emulsions, that is, small droplets ofhydrophilic fluid exist as individual compartments in a sea ofhydrophobic fluid. Droplets can be less than a micron in size (less thana femtoliter in volume) and an emulsion can have greater than 10¹⁰droplets per ml. Griffiths and Tawfik demonstrated that a gene librarydistributed in a cell-free extract and compartmentalized into dropletscan express their individual proteins in each droplet. In one case, theprotein is an enzyme that reacts with a substrate and the technique canbe used to evolve the enzyme with desired attributes. In another case,the gene is covalently bound to a bead that also contains an affinitymolecule that captures the gene product (e.g., using a protein tag),thereby associating the gene with its expression product for affinitymolecule selection. In addition, there is the technique noted above thatuses Hae III DNA methyltransferase.

Homogeneous noncompetitive immunoassays by definition do not requirephysical separation of an affinity molecule bound to its target beforedetection. A common example of this technique is aggregation oragglutination immunoassays. Another example is Förster (or fluorescence)resonance energy transfer (FRET), which is based on the transfer ofFörster energy (nonradiative transfer) from an excited fluorophore toanother fluorophore that is in proximity (Valanne et al. (2005) Anal.Chim. Acta 539, 251-6). A similar method uses a bioluminescent protein,such as luciferase, to excite a proximal fluorophore (BRET), typically afluorescent protein (Xu et al. (1999) Proc. Natl. Acad. Sci. USA 96(1),151-6). Another homogeneous assay alternative is a luminescentoxygen-channeling chemistry (Ullman et al. (1994) Proc. Natl. Acad. Sci.USA 91(12), 5426-30), wherein a light induced singlet oxygen generatingsystem transfers the singlet oxygen to a chemiluminescent system inproximity. The NanoDLSay system is a single-step homogeneous assay thatuses conjugated gold particles to form agregates in the presence of anantigen (Liu et al. (2008) J. Am. Chem. Soc. 130 (9), 2780-2). Proximityligation assay (PLA) uses two DNA single strands, one attached to eachaffinity molecule partner, that are complementary to a thirdoligonucleotide (Gullberg (2004) Proc Natl Acad Sci USA 101(22),8420-8424). When the affinity molecules are proximal to each other, thestrands hybridize to the linker oligonucleotide in an orientation whereends (3′ and 5′) are next to each other and can be ligated together. Theresulting DNA is amplified and quantified using Q-PCR.

Protein fragment compartmentalization (PFC) is similar to PLA in that 2complementary molecules are fused to potentially proximal binders thatinteract preferentially when in proximity. In this case, the moleculesare protein fragments capable of assembling into a complete andfunctional protein, typically an enzyme or fluorescent protein.Protein-protein interaction sensors using protein fragments were firstdeveloped by Nils Johnsson and Alexander Varshaysky using a splitubiquitin and this idea was further developed by Stephen Michnik in 1997(Pelletier et al. (1998) J. Biomol. Tech. acc. No. 50012) as an in vivoprotein-protein interaction analysis tool. The technique was used todevelop an in vivo antibody (scFv) screening method by fusing oneprotein fragment on the antigen and the other protein fragment on alibrary of scFv (Mössner et al. J. Mol. Biol. (2001) 308(2), 115-122;Koch et al. J. Mol. Biol. (2006) 357, 427-441; Secco et al. (2009) Prot.Evol. Des. Sel. 22(5), 149-158). Recently, Panbio Diagnostics hasdeveloped a homogeneous assay for the detection of antigen or antibodiesusing protein fragment complementation, which they call Forced EnzymeComplementation (FEC).

Most examples of affinity binder screening by PFC are in vivo, that is,the binding reactions are compartmentalized using cells. As mentionedabove, an alternative to using live cells is encapsulated cell-freeextracts using IVC, preferably manipulated using microfluidics. Whilethere are numerous examples of in vitro protein expression using IVC,only recently has this been done using microfluidic devices. Dittrich,et al. (Chembiochem. (2005) 6(5):811-4), has recently demonstrated invitro expression of a green fluorescent protein (red-shifted mutant) in5 micron (˜65 fL) microdroplets that were detected using confocalfluoroscopy. Few other researchers have developed this technology,preferring to use compartmentalized cell based assays (Brouzes et al.PNAS (2009) early edition).

SUMMARY

In some embodiments, the present invention provides a method forscreening specific affinity molecules to target molecules using ahomogeneous noncompetitive assay. In some embodiments, the methodcomprises use of reagents to perform a homogeneous non-competitiveassay, in which candidate affinity molecules are used to conduct thehomogeneous non-competitive assay in order to identify candidateaffinity molecules with affinity for the target as indicated by apositive result in the homogeneous non-competitive assay. In someembodiments, the affinity molecules are native antibodies, antibodyfragments, artificial antibody scaffolds, peptides, or nucleic acids. Insome embodiments, the native antibodies are IgG, IgM, IgA, or IgEmolecules; the antibody fragments include (Fab)₂, Fab, and scFv; and theartificial antibody scaffolds include Nanobodies, Affibodies,Anticalins, DARPins, Monobodies, Avimers, and Microbodies. In someembodiments, peptides are greater than three amino acids, consist ofeither natural or non-natural amino acids, and include peptide aptamers;the peptides are covalently attached to a carrier molecule. In someembodiments, the nucleic acid includes nucleic acid aptamers and peptidenucleic acids (PNA). In some embodiments, the affinity binders areexpressed from genes or chemically synthesized. In some embodiments, theaffinity molecules are comprised of a tyrosine/serine binary-codeinterface or a tyrosine/serine/X amino acid tertiary-code interface. Insome embodiments, two or more affinity molecules are required to bind toat least 2 different epitopes of a target molecule. In some embodiments,the binding of the first known affinity molecule and a second unknownaffinity molecule is an individual reaction performed in an individualvessel. In some embodiments, the individual vessel is a single reactiontube or a well of microtiter plate. In some embodiments, the individualvessels are water microdroplets, wherein water microdroplets can becreated by water-in-oil technology. In some embodiments, the watermicrodroplets are created using micro- or nanofluidic devices, wherein amicro- or nanofluidic device is used to manipulate microdroplets to mixreagents, perform reactions, heat, cool, detect and analyze assayoutput, and sort into various collection systems. In some embodiments,the reaction vessels are in vivo cells including bacteria,archaebacteria, fungal, insect, and mammalian cells.

In some embodiments, one affinity molecule is associated with a proteinfragment via a flexible linker that complements another protein fragmentassociated with the second affinity molecule via a flexible linker. Insome embodiments, complementation of the protein fragments associatedwith affinity molecules generates a measurable signal. In someembodiments, the measurable signal includes color, fluorescence, andbioluminescence. In some embodiments, the affinity molecules are inproximity when bound to the target to allow complementation ofassociated protein fragments. In some embodiments, one affinity moleculeis associated with a donor fluorophore via a linker that can transferForster energy to an acceptor fluorophore that is linked via a linker tothe second affinity molecule. In some embodiments, one affinity moleculeis associated with a bioluminescent protein via a linker that cantransfer Förster energy to an acceptor fluorophore that is linked via alinker to the second affinity molecule. In some embodiments, oneaffinity molecule is associated with a light induced singlet oxygengenerating system via a linker and the second affinity molecule is asinglet oxygen dependent chemiluminescent system (luminescent oxygenchanneling). In some embodiments, the affinity molecules are associatedwith gold particles conjugated with anti-epitope antibodies thataggregate when the reference affinity molecule and the unknown affinitymolecule (each with a different epitope tag) bind.

In some embodiments, the first affinity molecule is the referenceaffinity molecule and is known to bind the target with relatively highaffinity while the binding affinity of the second affinity molecule isnot known, but is determined by the homogeneous noncompetitive assay. Insome embodiments, the first affinity molecule has affinity for anepitope tag that is added to the target, wherein the epitope tag ispolypeptide expressed along with the protein affinity molecule,including, but not limited to, His-tag, FLAG-tag, V5-tag, HA-tag, andc-myc-tag. In some embodiments, the epitope tag is covalently bonded tothe target. In some embodiments, the second affinity molecule is derivedfrom a library of potential affinity molecules.

In some embodiments, the target molecule may be a protein, glycoprotein,phosphoprotein, other post-modification protein, protein complex,nucleic acid, protein:nucleic acid complex, carbohydrate, lipid complex,organic and inorganic molecule, including natural and synthetic versionsof any such molecules. The target or target molecules may comprise asingle protein or other biomolecule or multiple molecules (e.g., in amulti-molecular complex). For example, in some embodiments, affinitymolecules are used to simultaneously bind two or more molecules that arein proximity to one other, to, for example, detect such proximity.

Embodiments of the present invention further provide methods of usingthe complexes in therapeutic, diagnostic, and basic or applied researchsettings (e.g., drug screening applications).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary and detailed description is better understood whenread in conjunction with the accompanying drawings which are included byway of example and not by way of limitation.

FIG. 1 shows a cartoon representing a simple design of some embodiments,showing an affinity complex comprising a target and two affinitymolecules with attached complementary detection molecules. The target isrepresented by the spotted square with corner knockouts, which representepitopes of the target. The reference affinity molecule (oval withdownward diagonal) binds to one epitope and is associated to a detectionmolecule (oval with checkerboard). The unknown affinity molecule (ovalwith downward diagonal) binds to another epitope of the target and isassociated to a complementary detection molecule (oval with squares).The 2 complementary detection molecules function only when in proximity.

FIG. 2 shows an example of a microfluidic device. An immiscible fluid ispumped through the pathway of the device and an aqueous fluid containingan affinity molecule gene library in a cell-free translation solution isinjected forming microdroplets. Upon protein expression, the affinitymolecules bind to a target, the detection molecules are allowed tointeract, and detected in a sorting chamber. Positive samples are gatedto a collection bin while negative microdroplets are gated to the waste.

DEFINITIONS

As used herein, the term “about” means encompassing plus or minus 10%.For example, about 200 nucleotides refers to a range encompassingbetween 180 and 220 nucleotides.

As used herein, the term “gene” refers to a nucleic acid (e.g., DNA)sequence that comprises coding sequences necessary for the production ofa polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment is retained. Asused herein, the term “heterologous gene” refers to a gene that is notin its natural environment. For example, a heterologous gene includes agene from one species introduced into another species. A heterologousgene also includes a gene native to an organism that has been altered insome way (e.g., mutated, added in multiple copies, linked to non-nativeregulatory sequences, etc). Heterologous genes are distinguished fromendogenous genes in that the heterologous gene sequences are typicallyjoined to nucleic acid sequences that are not found naturally associatedwith the gene sequences in the chromosome or are associated withportions of the chromosome not found in nature (e.g., genes expressed inloci where the gene is not normally expressed).

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4 acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5(carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5 bromouracil,5-carboxymethylaminomethyl 2 thiouracil, 5carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6isopentenyladenine, 1 methyladenine, 1-methylpseudo-uracil, 1methylguanine, 1 methylinosine, 2,2-dimethyl-guanine, 2 methyladenine, 2methylguanine, 3-methyl-cytosine, 5 methylcytosine, N6 methyladenine, 7methylguanine, 5 methylaminomethyluracil, 5-methoxy-amino-methyl 2thiouracil, beta D mannosylqueosine, 5′ methoxycarbonylmethyluracil, 5methoxyuracil, 2 methylthio N6 isopentenyladenine, uracil 5 oxyaceticacid methylester, uracil 5 oxyacetic acid, oxybutoxosine, pseudouracil,queosine, 2 thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil, 4thiouracil, 5-methyluracil, N-uracil 5 oxyacetic acid methylester,uracil 5 oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6diaminopurine.

As used herein, the term “oligonucleotide” refers to a nucleic acid thatincludes at least two nucleic acid monomer units (e.g., nucleotides),typically more than three monomer units, and more typically greater thanten monomer units. The exact size of an oligonucleotide generallydepends on various factors, including the ultimate function or use ofthe oligonucleotide. To further illustrate, oligonucleotides aretypically less than 200 residues long (e.g., between 15 and 100),however, as used herein, the term is also intended to encompass longerpolynucleotide chains. Oligonucleotides are often referred to by theirlength. For example a 24 residue oligonucleotide is referred to as a“24-mer”. Typically, the nucleoside monomers are linked byphosphodiester bonds or analogs thereof, including phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like,including associated counterions, e.g., H⁺, NH₄ ⁺, Na⁺, and the like, ifsuch counterions are present. Further, oligonucleotides are typicallysingle-stranded. Oligonucleotides are optionally prepared by anysuitable method, including, but not limited to, isolation of an existingor natural sequence, DNA replication or amplification, reversetranscription, cloning and restriction digestion of appropriatesequences, or direct chemical synthesis by a method such as thephosphotriester method of Narang et al. (1979) Meth Enzymol. 68:90-99;the phosphodiester method of Brown et al. (1979) Meth Enzymol.68:109-151; the diethylphosphoramidite method of Beaucage et al. (1981)Tetrahedron Lett. 22:1859-1862; the triester method of Matteucci et al.(1981) J Am Chem Soc. 103:3185-3191; automated synthesis methods; or thesolid support method of U.S. Pat. No. 4,458,066, entitled “PROCESS FORPREPARING POLYNUCLEOTIDES,” issued Jul. 3, 1984 to Caruthers et al., orother methods known to those skilled in the art. All of these referencesare incorporated by reference.

The term “sample” is used in its broadest sense. In one sense it canrefer to an animal cell or tissue. In another sense, it is meant toinclude a specimen or culture obtained from any source, as well asbiological and environmental samples. Biological samples may be obtainedfrom plants or animals (including humans) and encompass fluids, solids,tissues, and gases. Environmental samples include environmental materialsuch as surface matter, soil, water, and industrial samples. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

A “sequence” of a biopolymer refers to the order and identity of monomerunits (e.g., nucleotides, etc.) in the biopolymer. The sequence (e.g.,base sequence) of a nucleic acid is typically read in the 5′ to 3′direction.

As used herein, the term “affinity complex” refers to an interactingmulticomponent collection of molecules that specifically interactsthrough interactions (e.g. hydrogen bonding, Van der Waals forces,electrostatic forces, hydrophobic forces, etc.) with a target molecule.

As used herein, the term “affinity molecule” refers to any molecule thatspecifically interacts through interactions (e.g. hydrogen bonding, Vander Waals forces, electrostatic forces, hydrophobic forces, etc.) with atarget molecule.

As used herein, the term “artificial antibody” or “antibody mimic”refers to any non-immunoglobulin molecule or molecular complex that iscreated to specifically interact with a target molecule.

As used herein, the term “epitope” refers to any surface region of atarget molecule to which an affinity molecule binds.

As used herein, the term “discontinuous epitopes” refers to two or moresurface regions of a target molecule or molecules that are separated bya defined distance.

The term “paratope” refers to the surface region of an affinity moleculethat interacts with the epitope of the target molecule.

As used herein, the term “affinity” refers to the non-random interactionof two molecules. The term “affinity” refers to the strength ofinteractions and can be expressed quantitatively as a dissociationconstant (K_(D)). One or both of the two molecules may be a peptide(e.g. antibody). Binding affinity (i.e., K_(D)) can be determined usingstandard techniques. For example, the affinity can be a measure of thestrength of the binding of an individual epitope with an antibodymolecule.

As used herein, the term “avidity” refers to the cooperative andsynergistic bonding of two or more molecules. “Avidity” refers to theoverall stability of the complex between two or more populations ofmolecules, that is, the functional combining strength of an interaction.

As used herein, the term “protein fragment complementation” refers to aprotein that can be fragmented into two or more parts so that when thefragments are in proximity they reform the original functional protein.

As used herein, the term “in vitro compartmentalization” refers to amethod of creating cell-like compartments using emulsion (water-in-oil)technology.

As used herein, the term “Förster resonance energy transfer” or “FRET”refers to a process in which energy is transferred between an excitedfluorophore (donor) and an acceptor fluorophore.

As used herein, the term “Bioluminescence resonance energy transfer” or“BRET” refers to a process in which energy is transferred between abioluminescent protein and an acceptor fluorophore.

DETAILED DESCRIPTION OF EMBODIMENTS

In some embodiments, the present invention provides a method forscreening specific affinity molecules to target molecules using ahomogeneous noncompetitive assay in a high throughput process. In someembodiments, the present invention provides compositions, systems, andmethods related to the screening of specific affinity molecules totarget molecules using a homogeneous noncompetitive assay in a highthroughput process. In some embodiments, the method comprises use ofreagents to perform a homogeneous non-competitive assay, in whichcandidate affinity molecules are used to conduct the homogeneousnon-competitive assay in order to identify candidate affinity moleculeswith affinity for the target as indicated by a positive result in thehomogeneous non-competitive assay.

In some embodiments, a target molecule contains two or more epitopes towhich the affinity binders can interact. In some embodiments, theepitopes are discontinuous. In some embodiments, the affinity moleculesrecognize the same epitopes. In some embodiments, the affinity moleculesrecognize different epitopes. In some embodiments, the affinitymolecules recognize multiplexed targets.

Affinity Molecules

In some embodiments, the affinity molecule comprises or consists of ascaffold that has a region known as a paratope or a target epitopeinteraction domain and a detection molecule connected via a linker. Insome embodiments, the paratope and detection molecule are situated toallow interaction with a target epitope and a freedom of the detectionmolecule. In some embodiments, each affinity molecule can comprise orconsist of the same scaffold. In some embodiments, each affinitymolecule can comprise or consist of different scaffolds.

In some embodiments, affinity molecules can be any antibody, antibodyfragment, scaffold or molecular construct that has a paratope domain orregion and a detection molecule domain or region. For example, IgGantibodies known to interact with a single target can used with amolecule that interacts with each Fc domain of the IgG (such as ProteinA or G) and contains the detection molecule. In some embodiments, Fabfragments of an IgG antibody are employed as the affinity molecule andcan be linked to the detection molecule through the constant domains (CLand CH1) of the molecule. In some embodiments, single chain fragments ofthe variable domains (scFv) are employed due to their increasedstability. In some embodiments, the smaller size of the VHH domain ofcamelids (Nanobodies) is a preferred affinity molecule.

In some embodiments, the affinity molecule is a monobody (fibronectintype III domain) derived from a human cell surface protein. Thisscaffold is structurally similar to antibody variable domains, but doesnot contain disulfide bonds that can hinder expression in prokaryoticsystems. In some embodiments, monobodies have a molecular weight of˜10,000 Daltons, they are very soluble, and thermally andproteolytically stable. In some embodiments, the monobody scaffoldcontains three loops (BC, DE, and FG loops) that can be collectivelyemployed as a paratope, similar to the CDR regions of an immunoglobulin.The polar opposite end of the paratope region contains three additionalloops (AB, CD, and EF loops). In some embodiments, the N-terminal,C-terminal, or AB, CD, and EF loops can be employed as a linker to thedetection molecule. In some embodiments, other linkers can be used, suchas an abbreviated rPEG.

In some embodiments, the affinity molecule is a DARPin (designed ankyrinrepeat protein) that is derived from a large class of repeat proteinsfound in various cellular sections in a variety of species. Each repeatconsists of 33 amino acid residues that form a beta-turn followed by twoanti-parallel helices and a randomized loop that is joined to thebeta-turn of the next repeat and functions to “stack” the repeatsgenerating a very stable hydrophobic core. In some embodiments, the loopand beta-turn sequences are involved in the paratope of the molecule. Insome embodiments, residues of the helices can contribute to theparatope. In some embodiments, the combination of the loop and beta-turnsequences and the residues of the helices generate a broad paratopeinterface. In some embodiments, three or more of these repeats arecreated to generate a molecule with very high affinity. In someembodiments, the ends of the repeats are “capped” to preserve thehydrophobic core, increase its solubility and stability, and can be usedfor labeling or immobilization. In some embodiments, N-terminal andC-terminal caps are employed as linkers to the detection molecule.

In some embodiments, the affinity molecule is an Affibody (the Z domainof Staphylococcal protein A) that comprises or consists of 58 aminoacids arranged as a bundle of 3 anti-parallel alpha helices. In someembodiments, the small size of the affibody molecule provides easierexpression and solubility in prokaryotic systems. In some embodiments,the affibody polypeptide is chemically synthesized then folded, whichallows the introduction of non-canonical amino acids in the interactiondomain or the addition of labels or reactive groups. In someembodiments, the interaction domain comprises or consists of 13 aminoacid residues that are randomized to generate a library from which anaffinity molecule is panned. In some embodiments, binding affinities foraffibodies and their substrates are in the nanomolar range.

In some embodiments, the affinity molecule is a Microbody (NascacellTechnologies). In some embodiments, a microbody is based on naturalcysteine-knot microproteins and cyclical knottins. In some embodiments,Microbodies are small (28 to 45 amino acids), yet very stable due tothree disulfide bonds within the structure, which allows the display ofa single peptide loop up to 20 amino acids. In some embodiments,microbodies are very soluble and are expressed from bacteria orsynthesized by chemical means then properly folded. In some embodiments,the stability and solubility of these proteins provides alternativetherapeutic delivery modes to the standard injection of mostbiologicals. In some embodiments, a very similar molecule called aVersabody (Amunix), acts as a primary affinity molecule. In someembodiments, a Versabody is a very small high disulfide density scaffoldbased on natural biopharmaceuticals, such as scorpion toxin. Versabodiesare extremely stable, soluble, and non-immunogenic.

In some embodiments, the affinity molecule is an Anticalin, an Avimer orthe domain A of an Avimer, a thioredoxin, an ubiquitin, agamma-crystallin, CTLA-4 (Evibody), or other recombinant artificialantibodies. In some embodiments, a primary affinity molecule is anymolecule capable of binding a target with a suitable affinity.

In some embodiments, the affinity molecule is a nucleic acid or peptideaptamer, wherein the nucleic acid or peptide contains a target affinitydomain and a detection molecule affinity domain.

Paratopes of Affinity Molecules

In some embodiments, the binding interaction of a paratope to itsepitope is based upon a combination of molecular contacts that togetheraccount for the affinity strength (e.g. Van der Waals interactions,hydrogen bonding, and hydrophobic interactions), specific amino acidside groups of the paratope polypeptide form bonds with amino acid sidegroups of the epitope polypeptide. In some embodiments, a portion of theamino acids in the paratope function as structural support. In someembodiments, antibody mimics have a single polypeptide paratope, such asAffibodies and Versabodies. In these embodiments, the sum of thoseinteractions determines the affinity. In some embodiments, affinitymolecules comprise multiple polypeptide loops or CDRs (complementaritydetermining regions), such as fibronectin Type III domains, ankyrinrepeats, and IgG molecules. These embodiments demonstrate additionalnumber and spacing of those interactions. In some embodiments, thestructure of the paratope should be adaptable to fit the epitope. Insome embodiments, the paratope has enough flexibility to form bonds withthe epitope without introducing intramolecular strain. In someembodiments, a large number of affinity molecules are be screened (e.g.,in a binding assay) to achieve a suitable structure.

In some embodiments, only moderate affinity interactions are required.In some embodiments, only moderate affinity interactions are preferred.In embodiments, increased effectiveness of screening libraries isachieved when moderate affinity is sought. In some embodiments, binary-or tertiary-code library systems reduce the size of the libraries,increase their effectiveness, and further simplify the process. In someembodiments, the basis of the binary-code interface within affinitymolecules is that effective affinity binders can be generated by usingonly 2 amino acids, tyrosine and serine (e.g. fibronectin type IIIdomains that were developed using the Tyr/Ser binary-code interfacedemonstrated affinities to 3 different proteins of 5 to 90 nM (Koide,A., et al., Proc. Nat. Acad. Sci. 104, 6632-6637, herein incorporated byreference in its entirety)). In some embodiments, a nanomolar affinitylevel, which can be achieved in binary-code interface, is very effectivein an affinity complex where the binding affinities are multiplied bythe linkage of the affinity molecules. In some embodiments, thecombination of a simplified binary-code interface library system and acooperative affinity complex system greatly reduces the time andresources necessary to development high affinity and specific affinitycomplexes.

Detection Molecules

In some embodiments, the detection molecule is a protein fragmentcomplementation system, wherein one protein fragment fused to oneaffinity molecule is complementary to another protein fragment fused tothe other affinity molecule and complementation of protein fragmentsgenerates a measurable signal (protein fragment complementation assay).In some embodiments, the complementary protein fragments generate anactive enzyme.

In some embodiments, the active enzyme is β-lactamase that can generatea colored product from a substrate such as nitrocefin, a fluorescentproduct from the substrate such as Fluorocillin Green, or abioluminescent product (in combination with firefly luciferase) from asubstrate such as Bluco (β-lactam-D-luciferin).

In some embodiments, the active enzyme is a luciferase that can generatebioluminescence from a substrate such as D-luciferin for fireflyluciferase and coelenterazine luciferin for renilla and gaussialuciferases (ref).

In some embodiments, the complementary protein fragments generate afluorophore such as green fluorescent protein, red fluorescent protein,or mutants of these proteins.

In some embodiments, the detection molecule is a donor or acceptorfluorophore that can be used in a Förster resonance energy transfer(FRET) assay. For example, one affinity molecule would be fused to thedonor fluorophore via a linker and the second affinity molecule would befused with the acceptor fluorophore via a linker. In some embodiments,the donor molecule is cyan fluorescent protein (CFP) and the acceptormolecule is yellow fluorescent protein. In some embodiments, the donormolecule is CyPet and the acceptor molecule is YPet. In someembodiments, the donor molecule is TagGFP and the acceptor molecule isTagRFP. In some embodiments, each affinity molecule fluorophore fusionprotein contains domains that have complimentary affinity, whereinproximal donor and acceptor fluorophores are spatially oriented to allowefficient energy transfer. In some embodiments, the complimentaryaffinity domains are leucine zipper or other coiled-coil domains. Insome embodiments, the complimentary affinity domains are affinitymolecules designed for expressly this purpose.

In some embodiments, the donor and/or acceptor fluorophore is a smallorganic or inorganic molecule that is fused to a polypeptide or othermolecule that has specific affinity for an expressed polypeptide fusedto the affinity molecule. For example, fluorescein isothiocyanate can beconjugated to the end of the K-coil of a coiled-coil dimer that iscomplementary to the E-coil that is fused to the affinity molecule as itis expressed. In some embodiments, the fluorophore is conjugated to achelated metal, such as nickel or copper, that binds a HisTag (4-10histidines) fused to the affinity molecule as it is expressed. In someembodiments, the fluorophore is conjugated to streptavidin that binds toa 15 amino acid Nanotag fused to the affinity molecule as it isexpressed.

In some embodiments, the fluorophore is conjugated to an affinitymolecule that has affinity for the expressed affinity molecule (that hasaffinity for the target). In some embodiments, the affinity molecule tothe target is fused with another affinity molecule that has affinity forthe fluorophore or a conjugated fluorophore. In some embodiments, theorganic fluorophore is a derivative of fluorescein, rhodamine, AlexaFluors (Invitrogen), CyDye Fluors (GE Healthcare Life Sciences), DyLightFluors (Dyomics GmbH), HiLyte Fluors (Anaspec) and the IRDye NearInfrared Fluors (Li-Cor).

In some embodiments, the inorganic fluorophore is a derivative of a rareearth metal chelate or cryptate (crown ether) such as lanthanium,terbium, samarium, dysprosium, or europium. In some embodiments, thefluorophore is a latex bead containing more than one fluorophore. Insome embodiments, the fluorophore is a phycobiliprotein, such asR-phycoerythrin or allophycocyanin. In some embodiments, the fluorophoreis a quantum dot.

In some embodiments, fluorophores are linked to either a NHS esterreactive group (reacts with ε-amine of lysine and the α-amine of thepolypeptide N-terminal) or a maleimide reactive group (reacts withreduced sulfhydryl of cysteine). In some embodiments, labeling proteinsnon-specifically, especially small polypeptides can potentiallyinterfere with their function. In some embodiments, it is important todemonstrate no loss of utility of the affinity molecule. In someembodiments, if the affinity molecule does not have a cysteine in thepolypeptide sequence (such as an Affibody or fibronectin scaffold), acysteine can be introduced at the C-terminal and specifically labeledwith any maleimide fluorophore.

In some embodiments, the FRET assay is time-resolved (TR-FRET), whereindetection of fluorescence of the acceptor fluorophore is determinedafter a short delay (for example, 100 μsec) after excitation of thedonor fluorophore, that is, the fluorescence of the donor has diminishedsignficantly and the lifetime of the acceptor is sufficiently extendedto measure its fluorescence.

In some embodiments, the donor detection molecule is a bioluminescentenzyme that can transfer resonance energy (BRET). For example, aluciferase enzyme typically generates light upon oxidation of itssubstrate, but can also transfer the energy to a fluorophore that is inproximity. In some embodiments, the bioluminescent enzyme is expressedas a fusion protein with one of the affinity molecules. In someembodiments, the bioluminescent protein is firefly, renilla, or gaussialuciferase. In some embodiments, the acceptor fluorophore is afluorescent protein that is fused to an affinity molecule. In someembodiments, the acceptor fluorophore is an organic or inorganic

In some embodiments, the bioluminescent enzyme is fused to a polypeptideor other molecule that has specific affinity for an expressedpolypeptide fused to the affinity molecule. For example, fireflyluciferase can be fused to the K-coil of a coiled-coil dimer that iscomplementary to the E-coil that is fused to the affinity molecule as itis expressed. In some embodiments, the bioluminescent enzyme isconjugated to a chelated metal, such as nickel or copper, that binds aHisTag (4-10 histidines) fused to the affinity moleculeas it isexpressed. In some embodiments, the bioluminescent enzyme is conjugatedor fused to an affinity molecule that has affinity for the expressedaffinity molecule (that has affinity for the target). In someembodiments, the affinity molecule to the target is fused with anotheraffinity molecule that has affinity for the bioluminescent enzyme. Insome embodiments, the acceptor fluorophore is protein expressed as afusion protein with one of the affinity molecules such as GFP, YFP, andRFP. In some embodiments, the acceptor fluorophore is an organicfluorophore, inorganic fluorophore, or quantum dot.

In some embodiments, the donor detection molecule is a light inducedsinglet oxygen generating system and the acceptor detection molecule isa chemiluminescent system that is excited by singlet oxygen (luminescentoxygen channeling).

In some embodiments, the detection system is a dynamic light scatteringassay, wherein the detection molecules are gold nanoparticles conjugateto affinity molecules with affinity for either reference of unknownaffinity molecule. For example, a portion of gold nanoparticles can beconjugated with anti-His tag antibodies and another portion withanti-FLAG antibodies. Aggregation occurs in the presence of the targetwhen both the affinity molecule expressing the His tag and the affinitymolecule expressing the FLAG tag bind the target.

In some embodiments, the detection molecule is covalently linked to theaffinity molecule via a flexible polymer such as a polypeptide (e.g.glycine/serine polypeptides), a nucleic acid strand, polyethyleneglycol, and peptide nucleic acid (PNA) that has sufficient degree offreedom to allow the interaction of the secondary affinity moleculeswith the primary affinity molecules. In some embodiments, the affinitymolecules are linked, either directly or linked via a suitable linker.The present invention is not limited to any particular linker group.Indeed, a variety of linker groups are contemplated, suitable linkerscould comprise, but are not limited to, alkyl groups, ether, polyether,alkyl amide linker, a peptide linker, a modified peptide linker, aPoly(ethylene glycol) (PEG) linker, a streptavidin-biotin oravidin-biotin linker, polyaminoacids (eg. polylysine), functionalisedPEG, polysaccharides, glycosaminoglycans, dendritic polymers such asdescribed in WO93/06868 and by Tomalia et al. in Angew. Chem. Int. Ed.Engl. 29:138-175 (1990), PEG-chelant polymers such as described inW94/08629, WO94/09056 and WO96/26754, oligonucleotide linker,phospholipid derivatives, alkenyl chains, alkynyl chains, disulfide, ora combination thereof.

In some embodiments the linker comprises a single chain connecting thedetection molecule to the affinity molecule. In some embodiments, thereare multiple linkers connecting the detection molecule to the affinitymolecule. In some embodiments, a linker may connect multiple thedetection molecules to the affinity molecule. In some embodiments, alinker attaches an additional functional portion to affinity molecule.In some embodiments, a linker may be branched, connecting more than twodetection molecules to the affinity molecule. In some embodiments, thelinker may be flexible, or rigid. In some embodiments, the linker of thepresent invention is cleavable or selectively cleavable. In someembodiments, the linker is cleavable under at least one set ofconditions, while not being substantially cleaved (e.g. approximately50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater remains uncleaved) underanother set (or other sets) of conditions. In some embodiments, thelinker is susceptible to enzymatic cleavage (e.g. proteolysis). In someembodiments, the enzymatic cleavage is site specific (e.g. sequencespecific). In some embodiments, the enzymatic cleavage is at a randomsite along the linker. In some embodiments, the enzymatic cleavage mayoccur at multiple random sites along the linker. In some embodiments,the linker is susceptible to cleavage under specific conditions relatingto pH, temperature, oxidation, reduction, UV exposure, exposure toradical oxygen species, chemical exposure, light exposure (e.g.photo-cleavable), etc.

In some embodiments, detection molecules are prepared by any suitablemethod. In some embodiments, IgG antibodies are used as affinitymolecules and can be linked via their reduced thiol groups, preferablyusing a crosslinking system from SoluLink. In some embodiments, one partof the anti-Fc IgG/Fab is labeled with MHPH(3-N-Maleimido-6-hydraziniumpyridine hydrochloride) and the other partwith MTFB (Maleimido trioxa-6-formyl benzamide). In some embodiments,the hydrazine moiety of the MHPH-modified molecules react with4-formylbenzamide of the MTFB-modified molecules to form stablebis-arylhydrazone-mediated conjugates. In some embodiments, alternativemethods for crosslinking proteins, known to those skilled in the art,are utilized. In some embodiments, an oligonucleotide can be synthesizedwith chemically reactive moieties (for example, a maleimide) on each endthat would react with the secondary affinity molecule. In someembodiments, each molecule could be conjugated to an oligonucleotide,one with a 3′ is free and the other with a 5′ free so that the twostrands can be ligated. In some embodiments, any suitable methods tolink the secondary affinity molecules would also be appropriate so longas the linker has flexibility to allow interaction of the secondaryaffinity molecules.

Target Molecules

In some embodiments, the target may be a protein, nucleic acid,carbohydrate, lipid, or other cell component. In some embodiments, theprotein may be native or denatured, modified (such as glyco- orphosphoproteins), part of a complex with other proteins, nucleic acids,or lipids (such as a lipid micelle), or part of a cell (or cell debris).In some embodiments, the nucleic acid may be DNA (single or doublestranded), RNA (message, ribosomal, transfer, transfer-message, smallinterfering, short hairpin, micro, piwi-interactive), or PNA (peptidenucleic acid). In some embodiments, the carbohydrate may be any numberof polysaccharides including glycogen, cellulose, and chitin. In someembodiments, the lipid may be a polyglyceride, wax, steroid, vitamin, orother natural hydrophobic molecules. In some embodiments, the target maybe native (natural) or recombinant, expressed, transcribed, orsynthesized, purified or part of a crude mixture, and with or without anepitope tag.

In some embodiments, the target may be a synthetic molecule, organic orinorganic, particle, or polymer.

Assay Screening

In some embodiments, the method of screening for an affinity moleculeincludes a target molecule, a reference affinity molecule that is knownto bind the target molecule with a known affinity, and an unknownaffinity molecule. In some embodiments, the reference affinity moleculehas affinity for a specific epitope on the target or an epitope tagadded to the target. In some embodiments, the coding sequence (eitherDNA or mRNA) for the reference affinity molecule (when it is a protein)is fused to the coding sequence of the detection molecule (when it is aprotein) and is expressed in the screening reaction using a cell-freetranslation. In some embodiments, fusions of affinity and detectionmolecules is expressed in a separate reaction, purified, and added tothe screening assay. In some embodiments, affinity and detectionmolecules are chemically conjugated in a separate reaction, purified,and added to the screening assay.

In some embodiments, the affinity molecule is fused or conjugated to asecondary affinity molecule that has affinity for the detection moleculeor the detection molecule may be fused or conjugated to a secondaryaffinity molecule that has affinity for the affinity molecule, and boththe affinity and detection molecules are added to the screening assay.For example, a monobody that has affinity for a target protein isexpressed with 10 histidine amino acids at the C-terminus and purified.This protein is mixed with a donor fluorophore conjugated to a nickelchelate complex that has affinity for the histidine tail and the entirecomplex is used in a FRET screening assay. This complex can be used foreither the reference affinity molecule or the unknown affinity molecule,but not both in the same reaction.

In some embodiments, the unknown affinity molecule is prepared in thesame way as the reference affinity molecule and added to the screeningassay containing the target and the reference affinity molecules in asingle reaction. In some embodiments, numerous unknown affinitymolecules are prepared and added to a multi-well plate containing thetarget and the reference affinity molecules in a multiplexed screeningassay. In some embodiments, a gene coding library of unknown affinitymolecules are mixed with the target molecule and the reference affinitymolecule (or the gene code for the reference affinity molecule) with itsdetection molecule in a cell-free extract capable of translating thegene library and the mixture is separated into microdroplets that areindividual reactions. In some embodiments, a microfluidic device is usedto create the microdroplets, merge and mix droplets, optimally heat thereactions (both translation and enzymatic assay if required), detect theoutput of the detection molecules (color, fluorescence, orbioluminescence light), sort and collect those droplets that exhibit apositive signal. In some embodiments, the microfluidic device amplifiesthe DNA in each droplet by polymerase chain reaction (PCR), or otheramplification techniques, creating sufficient DNA to identify the genesequence that codes for the positive binding reaction. In someembodiments, the microfluidic device (instrument) is a RainDanceTechnology instrument capable of creating, processing, and analyzing3000 droplets per second, which would allow the screening of over 10million reactions per hour (2.6×10⁸ per day).

Epitope Mapping

In some embodiments, a target molecule is mixed with variouscombinations of affinity molecules known to have affinity for the targetand, in association with their detection molecules, determined whichcombination produces a negative result indicating that both affinitymolecules have affinity for the same epitope. For example, a genelibrary that has been screened for affinity molecules to a targetmolecule generates 25 positive clones, each of which is PCR amplifiedwith both the detection molecule coding sequences and expressed incell-free extract to generate the specific affinity molecules fused witha reference detection molecule and its complement detection molecule.Affinity molecule #1 with its reference detection molecule is mixed withthe target molecule and placed in each well of the first row (24 wells)of a 384 well plate to which affinity molecule #2 with its complementarydetection molecule has been added to well A1, affinity molecule #3 withits complementary detection molecule has been added to well A2, and soon. The second row contains affinity molecule #2 with its referencedetection molecule, the third row with #3 and so on. Positive signalsindicate affinity binding of both affinity molecules to the target whilenegative signals indicate conflicting binding sites.

Systems and Kits

The present invention further provides systems and kits (e.g.,commercial therapeutic, diagnostic, or research products, reactionmixtures, etc.) that contain one or more or all components sufficient,necessary, or useful to practice any of the methods described herein.These systems and kits may include buffers, detection/imagingcomponents, positive/negative control reagents, instructions, software,hardware, packaging, or other desired components.

EXPERIMENTAL Example 1 Exemplary Use

The first example demonstrates homogeneous FRET analysis and itsimplementation in a microfluidic device. A small His tag labeled proteintarget is mixed with HiLyte Fluor™ 488 conjugated mouse anti-His tagmonoclonal antibody and a known anti-target antibody (IgG) labeled withHiLyte Fluor™ 555 in a cuvette tube and the fluorescence at ˜600 nmdetermined when exciting the solution at ˜500 nm (FRET). The samebinding reaction is loaded into a microfluidic device set up to measureFRET fluorescence in the same way. Next, each component is loaded intoindividual compartments of the microfluidic device, droplets created,merged, and mixed in line, and the FRET fluorescence determined.

Example 2 Exemplary Use of Protein Fragment Complementation

The second example demonstrates utility of complementary renilla proteinfragments fused to monobody affinity molecules via a peptide linker. Aprotein target is chosen for which the protein is available and themonobodies exist. The coding sequence for at least 2 monobodies iscloned into a cassette containing either the N-terminal renilla peptideor the C-terminal peptide, both using a ser/gly linker. The targetprotein, N-terminal renilla monobody, and C-terminal renilla monobody ismixed, added to renilla Luciferase Assay Reagent (Promega), and thebioluminescence measured using a luminometer. The same binding reactionis loaded into a microfluidic device set up to measure bioluminescencein the same way. Next, each component is loaded into individualcompartments of the microfluidic device, droplets created, merged, andmixed in line, and the bioluminescence determined. Finally, a genelibrary of C-terminal (or N-terminal) renilla monobodies coding forvarious peptide sequences at the BC and FG domains of the monobody isscreened for binding to the target using the known renilla monobody asthe reference.

Example 3 Exemplary Use of BRET

The third example demonstrates utility of a BRET system for screeningaffinity molecules. A protein target is chosen for which the protein isavailable and the monobodies exist. The coding sequence for one of themonobodies is cloned into a cassette containing a renilla luciferasesequence and the coding sequence for the other monobody is coned into acassette containing a red fluorescent protein (RFP). The target protein,renilla monobody, and RFP monobody is mixed, added to renilla LuciferaseAssay Reagent (Promega), and the fluorescence of the RFP measured usinga near infrared light detector. The same binding reaction is loaded intoa microfluidic device set up to measure bioluminescence in the same way.Next, each component is loaded into individual compartments of themicrofluidic device, droplets created, merged, and mixed in line, andthe bioluminescence determined. Finally, a gene library of C-terminal(or N-terminal) renilla monobodies coding for various peptide sequencesat the BC and FG domains of the monobody is screened for binding to thetarget using the known renilla monobody as the reference.

REFERENCES

The following references are herein incorporated by reference in theirentireties:

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1. A method for screening specific affinity molecules to targetmolecules using a homogeneous non-competitive assay, said methodcomprising: a) providing reagents of a homogeneous non-competitiveassay, b) combining candidate affinity molecules with said reagents toconduct said homogeneous non-competitive assay, and c) identifyingcandidate affinity molecules with affinity for said target as those thatproduce a positive result in said homogeneous non-competitive assay. 2.A method of claim 1, wherein said affinity molecules are nativeantibodies, antibody fragments, artificial antibody scaffolds, peptides,or nucleic acids.
 3. A method of claim 2, wherein said native antibodiesare IgG, IgM, IgA, or IgE molecules.
 4. A method of claim 2, whereinsaid antibody fragments include (Fab)₂, Fab, and scFv.
 5. A method ofclaim 2, wherein said artificial antibody scaffolds include Nanobodies,Affibodies, Anticalins, DARPins, Monobodies, Avimers, and Microbodies.6. A method of claim 2, wherein said peptides are greater than threeamino acids, consist of either natural or non-natural amino acids, andinclude peptide aptamers.
 7. A method in claim 6, wherein peptides arecovalently attached to a carrier molecule.
 8. A method of claim 2,wherein said nucleic acid includes nucleic acid aptamers and peptidenucleic acids (PNA).
 9. A method of claim 2, wherein said molecules areexpressed from genes or chemically synthesized.
 10. A method of claim 2,wherein said affinity molecules comprise a tyrosine/serine binary-codeinterface.
 11. A method of claim 2, wherein said affinity moleculescomprise a tyrosine/serine/X amino acid tertiary-code interface.
 12. Amethod of claim 1, wherein two or more affinity molecules are requiredto bind to at least 2 different epitopes of a target molecule.
 13. Amethod of claim 1, wherein the first affinity molecule is a referenceaffinity molecule and is known to bind the target with relatively highaffinity, wherein the binding affinity of the second affinity moleculeis not known, but is determined by the homogeneous assay.
 14. A methodof claim 13, wherein the reference affinity molecule has affinity for anepitope tag that is fused to the target.
 15. A method of claim 14,wherein the epitope tag is polypeptide expressed along with the proteinaffinity molecule, including His-tag, FLAG-tag, V5-tag, Nano-tag,HA-tag, and c-myc-tag.
 16. A method of claim 14, wherein the epitope tagis covalently bonded to the target.
 17. A method in claim 13, whereinthe unknown affinity molecule is derived from a library of potentialaffinity molecules.
 18. A method in claim 13, wherein the binding of thereference affinity molecule and an unknown affinity molecule is anindividual reaction.
 19. A method in claim 13, wherein the bindingreactions are performed in individual vessels.
 20. A method in claim 13,wherein the individual vessels are reactions tubes.
 21. A method inclaim 13, wherein the individual vessels are part of microtiter plates.22. A method in claim 13, wherein the individual vessels are aqueousmicrodroplets.
 23. A method in claim 22, wherein aqueous microdropletsare created by water-in-oil technology.
 24. A method in claim 22,wherein the water microdroplets are created using micro- ornanofluidics.
 25. A method in claim 23, wherein a micro- or nanofluidicdevice is used to manipulate microdroplets to mix reagents, performreactions, heat, cool, detect and analyze the output of the homogeneousnoncompetitive assay, and sort into a collection system.
 26. A method inclaim 18, wherein the reaction vessels are in vivo cells.
 27. A methodin claim 26, wherein cells include bacteria, archaebacteria, fungal,insect, and mammalian cells.
 28. A method of claim 1, wherein thehomogeneous noncompetitive assay is a protein fragment complementationassay (PFCA).
 29. A method of claim 13, wherein one affinity molecule isassociated with a protein fragment via a flexible linker thatcomplements another protein fragment associated with the second affinitymolecule via a flexible linker.
 30. A method of claim 14, whereincomplementation of protein fragments associated with affinity moleculesgenerates a measurable signal.
 31. A method of claim 15, wherein ameasurable signal includes color, fluorescence, or bioluminescence. 32.A method of claim 1, wherein the homogeneous noncompetitive assay is aFörster resonance energy transfer (FRET) assay.
 33. A method of claim32, wherein the FRET assay is time-resolved FRET.
 34. A method of claim32, wherein one affinity molecule is associated with the donorfluorophore via a linker and the second affinity molecule is associatedwith the acceptor fluorophore via a linker.
 35. A method of claim 34,wherein the fluorophores for both affinity molecules are fluorescentproteins that are expressed as a fusion to the affinity moleculeprotein.
 36. A method in claim 35, wherein the fluorescent proteins arematched FRET pairs, including cyan fluorescent protein (CFP):yellowfluorescent protein, CyPet:YPet and TagGFP:TagRFP.
 37. A method of claim34, wherein the fluorophore for the affinity molecule that is known tobind the target with relatively high affinity is covalently conjugatedto the binder.
 38. A method of claim 1, wherein the homogeneousnoncompetitive assay is a bioluminescence resonance energy transfer(BRET) assay.
 39. A method of claim 38, wherein the bioluminescentprotein expressed as a fusion protein with one of the affinity moleculesincludes firefly luciferase, renilla luciferase, and gaussia luciferase.40. A method of claim 38, wherein the fluorophore protein expressed as afusion protein with one of the affinity molecules includes GFP, YFP, andRFP.
 41. A method of claim 1, wherein the homogeneous noncompetitiveassay is an enzyme channeling assay.
 42. A method of claim 41, whereinthe enzyme channeling assay is a luminescent oxygen channelingimmunoassay (LOCI).
 43. A method of claim 42, wherein the luminescentoxygen channeling immunoassay is the AlphaLISA system.
 44. A method ofclaim 1, wherein the homogeneous noncompetitive assay is a dynamic lightscattering assay.
 45. A method of claim 44, wherein the dynamic lightscattering assay uses conjugated gold particles.
 46. A method of claim45, wherein the dynamic light scattering assay is the NanoDLSay system.47. A method in claim 1, wherein the target molecule may be a protein,glycoprotein, phosphoprotein, other post-modification protein, proteincomplex, nucleic acid, protein:nucleic acid complex, carbohydrate, lipidcomplex, organic and inorganic molecule.